RESUMO
Identification of dynamic protein-protein interactions at the peptide level on a proteomic scale is a challenging approach that is still in its infancy. We have developed a system to cross-link cells directly in culture with the special lysine cross-linker bis(succinimidyl)-3-azidomethyl-glutarate (BAMG). We used the Gram-positive model bacterium Bacillus subtilis as an exemplar system. Within 5 min extensive intracellular cross-linking was detected, while intracellular cross-linking in a Gram-negative species, Escherichia coli, was still undetectable after 30 min, in agreement with the low permeability in this organism for lipophilic compounds like BAMG. We were able to identify 82 unique interprotein cross-linked peptides with <1% false discovery rate by mass spectrometry and genome-wide database searching. Nearly 60% of the interprotein cross-links occur in assemblies involved in transcription and translation. Several of these interactions are new, and we identified a binding site between the δ and ß' subunit of RNA polymerase close to the downstream DNA channel, providing a clue into how δ might regulate promoter selectivity and promote RNA polymerase recycling. Our methodology opens new avenues to investigate the functional dynamic organization of complex protein assemblies involved in bacterial growth. Data are available via ProteomeXchange with identifier PXD006287.
Assuntos
Bacillus subtilis/metabolismo , Proteínas de Bactérias/metabolismo , Glutaratos/química , Mapeamento de Interação de Proteínas/métodos , Succinimidas/química , Sequência de Aminoácidos , Bacillus subtilis/química , Bacillus subtilis/genética , Proteínas de Bactérias/química , Proteínas de Bactérias/genética , Reagentes de Ligações Cruzadas/química , Meios de Cultura/química , DNA Bacteriano/química , DNA Bacteriano/genética , DNA Bacteriano/metabolismo , RNA Polimerases Dirigidas por DNA/química , RNA Polimerases Dirigidas por DNA/genética , RNA Polimerases Dirigidas por DNA/metabolismo , Escherichia coli/química , Escherichia coli/genética , Escherichia coli/metabolismo , Expressão Gênica , Glutamato Desidrogenase/química , Glutamato Desidrogenase/genética , Glutamato Desidrogenase/metabolismo , Biogênese de Organelas , Ligação Proteica , Subunidades Proteicas/química , Subunidades Proteicas/genética , Subunidades Proteicas/metabolismo , Ribossomos/genética , Ribossomos/metabolismo , Especificidade da Espécie , Fatores de Elongação da Transcrição/química , Fatores de Elongação da Transcrição/genética , Fatores de Elongação da Transcrição/metabolismoRESUMO
PHB granules are bacterial organelles that store excess carbohydrates in the form of water-insoluble polyhydroxybutyrate (PHB). The PHB polymerase, phasin (a small amphipathic protein), and active PHB synthesis are essential for the formation of mature PHB granules in Caulobacter crescentus. Granule formation was found to be initiated by the condensation of self-associating PHB polymerase-GFP into foci, closely followed by the recruitment and condensation of phasin-mCherry. Following the active synthesis of PHB and granule maturation, the polymerase dissociates from mature granules and the PHB depolymerase is recruited to the granule. The polymerase directly binds phasin in vitro through its intrinsically disordered N-terminal domain. Thus, granule biogenesis is initiated and controlled by the action of a PHB polymerase and an associated helper protein, phasin, that together synthesize the hydrophobic granule's content while forming the granules protein boundary.